A quantitative analysis of coconut water: a new storage media

for avulsed teeth

Velayutham Gopikrishna, MDS,a Toby Thomas, MDS,b and
Deivanayagam Kandaswamy, MDS,c Chennai, India
MEENAKSHI AMMAL DENTAL COLLEGE

Objective. The purpose of this study was to use a Collagenase-Dispase assay to investigate the potential of a new
storage media, coconut water, in maintaining viable periodontal ligament (PDL) cells on simulated avulsed teeth.
Study design. Fifty freshly extracted human teeth were divided into 3 experimental groups and 2 control groups. The
positive and negative controls corresponded to 0 minutes and an 8-hour dry time, respectively. The experimental teeth
were stored dry for 30 minutes and then immersed in 1 of the 3 media: coconut water (CW), Hank’s balanced salt
solution (HBSS), and milk. The teeth were then treated with Dispase grade II and Collagenase for 30 minutes. The
number of viable PDL cells were counted with a hemocytometer and analyzed.
Results. Statistical analysis demonstrated that CW kept significantly more PDL cells viable compared to either HBSS or
milk.
Conclusion. Within the parameters of this study, it appears that CW may be better alternative to HBSS or milk in
terms of maintaining PDL cell viability after avulsion and storage. (Oral Surg Oral Med Oral Pathol Oral Radiol
Endod 2008;105:e61-e65)

Clinical surveys indicate that traumatic dental injuries
in children and adolescents are a common problem and
studies have shown that prevalence of these injuries is
increasing.1 Avulsion injury, one of the most severe
forms of dental trauma, is characterized by complete
displacement of the tooth from its alveolar socket.
Because of the complexity of this injury, the neurovas-
cular supply is severely compromised and usually re-
sults in loss of pulp vitality.
In cases of tooth avulsions, the primary goal is to
preserve the vitality of the periodontal ligament (PDL)
cells attached to the root surface until appropriate treat-
ment can be performed. This may bring about a favor-
able reattachment of the periodontal ligament. Soder
et al.2 showed that after avulsion, the number of viable
This study was supported by a scientific research grant given by the
Meenakshi University, Chennai, India, vide Grant No MAHER/517/
2006. The authors thank M. Parthiban, PhD, Assistant Professor,
Department of Animal Biotechnology, Tamilnadu University of Vet-
erinary and Animal Sciences (TANUVAS), Chennai, India for his
technical and collaborative support.
a
Reader, Department of Conservative Dentistry and Endodontics,
Meenakshi Ammal Dental College, Chennai, India.
b nesium), and sugars lost from the body during heavy
Lecturer, Department of Conservative Dentistry and Endodontics,
Meenakshi Ammal Dental College, Chennai, India.
c
Professor and Head, Department of Conservative Dentistry and
Endodontics, Meenakshi Ammal Dental College, Chennai, India.
Received for publication Mar 26, 2007; returned for revision Jul 20,
2007; accepted for publication Aug 1, 2007.
1079-2104/$ - see front matter
© 2008 Mosby, Inc. All rights reserved.
doi:10.1016/j.tripleo.2007.08.003
cells on the root surface decreased with increased dry-
ing time and that after 2 hours it was not possible to
demonstrate cell viability. Therefore, the ideal treat-
ment of choice at the time of avulsion is immediate
replantation, thus reestablishing the natural nutrient
supply to the periodontal ligament cells, thereby mini-
mizing further damage and enhancing the healing pro-
cess. Unfortunately, some situations may occur that
delay immediate replantation. Where such situations
exist, the tooth should be stored in a medium that
maintains periodontal ligament cell viability until de-
finitive dental treatment can be accomplished.
The ability of a storage/transport medium to support
cell viability can be more important than the extraoral
time to prevent ankylosis and replacement resorp-
tion.2,3 Various storage media such as tap water, saliva,
saline, milk, culture media, Viaspan, and Hank’s Bal-
anced Salt Solution (HBSS) (marketed as Save-A-
Tooth) have been employed. Dumsha4 and Patil et al.5
suggested storing the avulsed tooth in milk, HBSS, or
saline.
The biologically pure, tender coconut water helps to
replace fluids, electrolytes (potassium, calcium, and mag-
physical exercise. It is used as a blood plasma substitute
as it is sterile and readily accepted by the body.6 Taking
these properties into consideration we hypothesized
that this natural isotonic drink could be a viable storage
medium in the transportation of an avulsed tooth. Cur-
rently no studies have suggested using coconut water as
a storage medium.

e61

e62 Gopikrishna et al.
Several techniques have been used to determine the
viability of the PDL cells following avulsion; however,
most of the experimental data that are available have
been obtained using techniques in which the cells are
cultured and/or trypsinized for longer periods of time.
As the extracellular matrix has a high content of colla-
gen and other proteins, it seems reasonable that the use
of enzymatic desegregation would provide a greater num-
ber of cells within a shorter time frame.7 Pileggi et al.8
used a collagenase dispase assay to compare PDL via-
bility of simulated avulsed teeth after storage in HBSS,
milk, saline, or water. Both collagenase and dispase
enzymes disrupt the extracellular matrix and cause the
release of cells without excessive disruption and de-
struction of their own membrane. Therefore, these au-
thors suggested that the use of these 2 enzymes may
provide additional data regarding the viability of PDL
cells after avulsion injury. Furthermore, this method may
be more representative of the actual clinical situation
because the cells are not subjected to long processing
times to determine their viability status.
The purpose of this study was to evaluate the efficacy
of a new storage medium, coconut water, in comparison
with other traditional storage media like HBSS and
milk in maintaining the viability of PDL cells using a
collagenase-dispase assay.
MATERIALS AND METHODS
Fifty-five human teeth with closed apices that were
extracted for orthodontic purpose were obtained for this
study. The average age of the patient was 24 years.
Teeth extracted from patients with moderate to severe
periodontal disease or with extensive caries were ex-
cluded. Extractions were performed as atraumatically
as possible by an oral surgeon. Following extractions,
the teeth were held with forceps by the coronal region,
and the coronal 3 mm of PDL was scraped with a
curette to remove cells that may have been damaged.
The teeth were then randomly divided into 1 of the 3
experimental storage solution groups: group 1, coconut
water; group 2, HBSS; and group 3, milk. There were
15 samples per group. The positive and the negative
control group consisted of 5 samples each. The teeth in
the experimental groups were dried for 30 minutes
(during which time the coronal PDL cells were curet-
ted), followed by a 45-minute immersion in 1 of the 3
storage solution groups. The positive control teeth were
neither dried nor were they stored in any solution, but
rather they were immediately treated with dispase and
collagenase. The negative control teeth were bench-
dried for 8 hours, with no follow-up storage solution
time, and then placed in the dispase and collagenase.
Each experimental tooth, after drying and soaking,
was incubated for 30 minutes in 15-mL Falcon tubes
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February 2008
Table I. Number of viable cells (M ⫾ SD) for the
various test groups
Number of Mean number of
Groups samples viable cells SD
CW 15 525.00 9.51
HBSS 15 446.73 19.12
MLK 15 185.60 12.01
PC 5 3762.729 426.018
NC 5 38.700 5.16
CW, coconut water; HBSS, Hank’s balanced salt solution; MLK, milk;
PC, positive control; NC, negative control.
with a 2.5-mL solution of 0.2 mg/mL–1 of collagenase
CLS II (Cooper Biomedical, Malvern, PA) and a 2.4
mg/mL–1 solution of dispase grade II (Gibco, Taastrup,
Denmark) in phosphate-buffered saline (PBS). After
incubation, 50 ␮L of fetal bovine serum (FBS) was
added to each tube. All tubes were then centrifuged for
4 minutes at 110 ⫻ g. The supernatant was then re-
moved with sterile micropipettes, and the cells were
labeled with 0.4% Trypan Blue for determination of
viability, according to Polverini and Leibovich.9 The
number of viable protective least significant difference
(PDL) cells was counted under a light microscope with
a hemocytometer at ⫻20 magnification. Statistical
analysis of the results was done by using Tukey-HSD
multiple range test.
RESULTS
The teeth stored in coconut water demonstrated sig-
nificantly the highest number of viable PDL cells fol-
lowed in rank order by HBSS and milk.
There was also a significant difference in the number
of viable PDL cells between HBSS and milk. All ex-
perimental solution groups were significantly lower
than positive control and higher than negative control
groups (Table I).
DISCUSSION
Avulsion is a traumatic injury leading to loss of
attachment of periodontal ligament from the alveolar
socket. The treatment of choice in such cases is to
replant the tooth back into the socket. The importance
of PDL cell viability before reimplantation was dem-
onstrated by Hammer to prove that the length of sur-
vival of a reimplanted tooth is directly correlated with
amount of viable periodontal membrane.10 One of the
sequelae following replantation of an avulsed tooth
includes inflammatory or replacement resorption.11 The
development of inflammatory root resorption is directly
related to damage of the periodontium at the time of the
accident and the presence of bacteria within the root
canals and dentinal tubules. The development of re-
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Volume 105, Number 2
placement resorption depends on both the degree of
damage to the periodontium at the time of avulsion12-14
and the extent to which the viability of periodontal
ligament cells remaining on the tooth surface is main-
tained.15 Hence, the prognosis of an avulsed tooth is
largely dependent on the status of the periodontal lig-
ament cells at replantation. Therefore, the predominant
philosophy derived from the research of Andreasen and
Hjørting-Hansen for the treatment of avulsed teeth is:
Replant the tooth immediately or as quickly as possible
after the avulsion.16 But, certain situations, like pres-
ence of more severe injuries needing immediate medi-
cal attention or nonavailability of a dental hospital close
by can lead to a delay in immediate replantation of the
avulsed tooth. In such situations the teeth should be
stored in a medium that can maintain the periodontal
ligament cell viability until definite dental treatment is
accomplished.
According to Andreasen and Hjørting-Hansen,16
teeth that were replanted quickly (within 30 minutes)
had a better success rate than those that were extraoral
for longer periods of time before replantation. In the
current investigation, a 30-minute dry time was chosen,
as this seems to be a critical time at which damage has
been done to many PDL cells, yet some cells remain for
assessment. Also, 30 minutes represents a typical clin-
ical scenario during which the avulsed tooth may re-
main dry before being placed into a storage medium.
Some experimental studies have indicated that stor-
age media is a more critical prognostic factor than the
extra-alveolar period.2,17-19 Physiologic storage media
such as milk, saliva, saline, HBSS, Propolis, and Vias-
pan have been used for preservation of viability of
periodontal ligament cells.8,20-22
Blomlof et al.15,18 has shown storage of avulsed teeth
in tap water and saliva to be damaging to periodontal
ligament cells causing increased root resorption.23,24
Cvek et al.25 found that avulsed teeth that were soaked
in an isotonic saline solution for 30 minutes before
replantation showed less resorption than those that were
stored dry for 15 and 40 minutes. Recent studies have
evaluated the use of 0.9% isotonic saline, milk, HBSS,
and Viaspan as storage media for the preservation of
cell viability.20-22 HBSS was the most effective,20,22
although milk and saline were suitable, provided the
extraoral time did not exceed 2 hours.21
In the dental literature, various techniques have
been used to quantitate the number of viable PDL
cells. Reinholdt et al.26 used a stepwise trypsiniza-
tion procedure by exposing samples to trypsin 3
consecutive times for 20 minutes each. Soder et al.27
used chromogenic stain to quantitate viable PDL cells.
Patil et al.28 used a stepwise trypsinization procedure
and fluorescein diacetate as a new staining technique
Gopikrishna et al. e63
for determining the viability of PDL cells in simulated
avulsion injuries.
In the current study, to minimize the exposure of
cells to active trypsin and to preserve maximum cell
viability, the root surface was treated with collagenase
and dispase grade II as was performed in the work by
Pileggi et al.29 This procedure allowed rapid cell re-
trieval and maintained maximum cellular integrity, as
was demonstrated by the positive control samples.
Coconut water has never been tested for its potential
benefits on PDL cells of avulsed teeth. This study
compared coconut water, HBSS, and milk in terms of
PDL cell viability. The coconut water group demon-
strated significantly more viable PDL cells than HBSS
and milk, with the HBSS group showing significantly
more viable PDL cells than milk.
Coconut (Cocos nucifera L.), popularly known as
“Tree of Life,” is a natural drink produced biologically
and hermetically packed inside the coconut in a hy-
gienic way without any contamination. The electrolyte
composition of coconut water resembles intracellular
fluid more closely than extracellular plasma. The pre-
dominant cations are potassium, calcium, and magne-
sium. Sodium, chloride, and phosphate are found in
much lower concentrations. It is a hypotonic solution
that is more acidic than plasma, and has a specific
gravity of approximately 1.020, comparable with blood
plasma.30
Coconut water has a high osmolarity because of the
sugars present, which are primarily glucose and fruc-
tose. It is also rich in many essential amino acids
including lysine, cystine, phenylalanine, histidine, and
tryptophan.30 Coconut water is readily accepted by the
human body and is a safe means of rehydration particu-
larly in patients suffering from potassium deficiency.31 In
fact, coconut water has been shown to be just as effec-
tive as commercial electrolyte solutions in prolonging
survival time in sick patients.32
Superior maintenance of viability of the PDL cells in
the coconut water group may be due to the nutrients
that are present in coconut water such as proteins,
amino acids, vitamins, and minerals, which help in
nourishing the cells and maintaining their viability (Ta-
ble II). In a study conducted by Majundar,33 coconut
water was found to be sterile and nonhemolytic.
Edirweera6 used coconut water intravenously as a sub-
stitute for normal saline.
Blomlof15 showed that the important factor in main-
taining the viability is the osmolarity of the transport
medium. Andreasen17 found that, in contrast to tap
water, both physiologic saline and saliva, with their
differences in chemical composition but similar osmo-
lality, were able to decrease the incidence of root re-
sorption. This suggests that the viability of the PDL is
 OOOOE
e64 Gopikrishna et al. February 2008

Table II. Composition of tender coconut water

Minerals, mg/100 mL Amino acids, % of total protein Vitamins, value in ␮g/mL
Potassium 291 Alanine 2.41 Nicotinic acid 0.64
Sodium 42 Arginine 10.75 Pantothenic acid 0.52
Chlorine 75 Aspartic acid 3.60 Biotin 0.02
Calcium 44 Cystine 0.97-1.17 Riboflavin 0.01
Magnesium 10 Glutamic acid 9.76-14.5 Folic acid 0.003
Sulphur 58 Histidine 1.95-2.05 Thiamine Trace
Phosphorus 9.20 Leucine 1.95-4.18 Pyridoxine Trace
Iron (mg/100g) 06 Lysine 1.95-4.57
Copper 26 Proline 1.21-4.12
Phenylalanine 1.23
Serine 0.59-0.91
Tyrosine 2.83-3.00

more closely linked to the osmolarity of the solution
than to its chemical composition. The water permeabil-
ity of a cell is high, and therefore, in a hypotonic
solution, the cells will swell and rupture, whereas in a
hypertonic solution they will shrink because of water
movement out of the cell. It has been reported that cell
growth can occur at a range of 230 to 400 mOsm/L.34
When measured in an osmometer, the osmolarity of
the HBSS, milk, and coconut water was found to be
295 mOsm/L, 232 mOsm/L, and 372 mOsm/L, re-
spectively. Blomlof15 found that HBSS was slightly
better for maintaining the cell integrity than milk, sa-
liva, or saline. This was mainly because of its physio-
logic osmolarity. Milk was found to have the lowest
osmolarity among the 3 but also within the range, but
the inability of the cells to multiply and divide might be
the reason that there was a drastic decrease in number
of viable cells. The osmolarity of coconut water was
found to be to be the highest and this might have
enabled the superior maintenance of cell viability.
CONCLUSION
Within the parameters of this study, it appears that
the ability of coconut water to maintain PDL cell via-
bility is statistically superior to HBSS and milk.
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